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(Received for publication, August 30, 1994; and in revised form, December 12, 1994)
The mannose transporter complex acts by a mechanism which
couples translocation with phosphorylation of the substrate. It
consists of a hydrophilic subunit (IIAB
) and two
transmembrane subunits (IIC, IID). The
purified complex was reconstituted into phospholipid vesicles by octyl
glucoside dilution. Glucose export was measured with proteoliposomes
which were loaded with radiolabeled glucose and to which purified
IIAB, cytoplasmic phosphorylcarrier proteins, and
P-enolpyruvate were added from the outside. Vectorial transport was
accompanied by stoichiometric phosphorylation of the transported sugar.
Glucose added to the outside of the proteoliposomes was also
phosphorylated rapidly but did not compete with vectorial export and
phosphorylation of internal glucose. Glucose uptake was measured with
proteoliposomes which were loaded with the cytoplasmic phosphoryl
carrier proteins and P-enolpyruvate and to which glucose was added from
the outside. Vectorial import and phosphorylation occurred with a
higher specificity (K
30 ± 6
µM, k
401 ± 32 pmol of
Glc/µg of IICD/min) than nonvectorial phosphorylation (K 201 ± 43 µM, k 975 ± 88 pmol of Glc/µg of
IICD/min).
A new plasmid pTSHIC9 for the controlled
overexpression of the cytoplasmic phosphoryl carrier proteins, enzyme
I, HPr, and IIA
, and a simplified procedure for the
purification of these proteins are also described.
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